Biology Reference
In-Depth Information
2.3 Human AB Serum and M-CSF
2.3.1 AB Serum
Human peripheral blood monocytes can be differentiated to macrophages in culture
in human AB serum, and enriched through their adherence to tissue culture plates.
Initially, the monocytes contain mostly PDE4 activity, with some PDE3 activity. As
the cells differentiate in culture, the predominate PDEs emerge as PDE1 and PDE3,
with rapidly declining amounts of PDE4 activity. PDE2 and PDE5 levels remain
consistently low throughout the differentiation process (Gantner et al.
1997a
). In
another study with human monocytes, the majority of the PDE4 activity was found
in the soluble fraction, and the PDE3 activity was associated with the particulate
fraction similar to its location in many other tissues (Verghese et al.
1995
). AB
serum-differentiated monocytes exhibit a PDE profile strongly resembling that
observed for human alveolar macrophages. A comparison of TNF-
a
production
elicited by lipopolysaccharide (LPS) between monocytes and monocyte-derived
macrophages showed inhibition of TNF-
a
production with increases in cAMP
brought on by PDE4 inhibition in both cell types (Gantner et al.
1997a
). PDE4
controlled the largest portion of TNF-
a
production in monocytes, as about 80%
could be blocked with a PDE4 inhibitor, with PDE3 inhibition decreasing TNF-
a
production approximately 10%. In macrophages, PDE3 and PDE4 controlled more
equal amounts, but PDE inhibition was only effective when administered in the
presence of an additional cAMP stimulus, perhaps indicating a lower cAMP tone in
macrophages than monocytes.
Human monocytes cultured in human AB serum also have a very similar PDE
profile to that present in the cell line Mono-Mac-6. Using this cell line as a
surrogate, a closer look at the changes in the PDE4 isoform levels during challenge
with cAMP-increasing agents showed that db-cAMP, PGE
1
, or LPS transiently
increased PDE4A, B, and D mRNA and protein levels (Verghese et al.
1995
).
However, the time course and magnitude of induction of each isoform was not
identical. PDE4B levels rose early, mRNA increased from 2 to 4 h, protein
increased from 2 to 8 h; with PDE4A and PDE4D rising later in the time course,
mRNA increased from 2 to 8 h and protein increased after 5-24 h of treatment. This
increase was specific for PDE4 isoforms as there was no concomitant increase in
PDE3 activity with differentiation. An additional level of regulation was observed
for the PDE4 isoforms as differences in the induction of mRNA when compared to
protein levels were apparent over the time courses tested, suggesting that the PDE4
isoforms and splice variants were transcriptionally regulated.
2.3.2 M-CSF
รพ
AB Serum
While PDE activities, and therefore the duration of cAMP and cGMP signals, can
change during differentiation, the levels of their downstream signaling effector
